CN109593707A - 一种羊水细胞培养基及其制备方法 - Google Patents
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Abstract
本发明公开了一种羊水细胞培养基及其制备方法，包括基础培养基以及添加在所述基础培养基中的添加成分，以基础培养基的体积作为基准，所述添加成分组成及其含量分别为结合蛋白，10‑15mg/L、转铁蛋白，10‑15mg/L、胰岛素，15‑20mg/L、L‑谷氨酰胺，2‑4mM、大豆胰酶，2‑4mg/L、生长因子，5‑15ug/L、孕酮，1‑2nmol/L、氢化皮质酮，2‑3nmol/L、葡萄糖，5‑25mM、脂类复合物，2‑4mM以及亚硒酸钠，20‑40nmol/L。本发明采用无血清等动物源的成分，排除血清中含有许多不确定物质对于羊水细胞体外培养造成的不利影响，此外添加有维生素E和L‑抗坏血酸作为抗氧化剂能够促进羊水细胞的成长，具有协同效应，使得羊水细胞体外培养过程中克隆总数以及增殖速率等均有一定的提高，进而能够大大缩短羊水细胞的培养周期。
Description
技术领域
发明涉及细胞体外培养技术领域，具体为一种羊水细胞培养基及其制备方法。
背景技术
羊水细胞比其它普通类型的细胞有着很多优点，首先，正常情况下羊水细胞在孕妇临产前就能够被提取，并可以从这些羊水细胞中建立大量的iPS细胞，医生通过对这些被重组的羊水细胞进行检查来判断孕妇是否患有疾病；其次，羊水混合物中包含有各种类型胎儿的体内细胞。由于羊水混合物中所包含细胞的“年龄”都比较小，因此这些细胞遭受周围环境引起的诱发突变的可能性就很小，从遗传学角度来说它们会更稳定。
但是，由于羊水中活细胞少，培养周期长，无菌要求高，稍不注意即致失败。即使培养成功，因分裂相少，形态不佳，达不到分析要求，无法进行进一步研究。因此，成功的羊水细胞培养，除需严格的实验操作技术和经验外，培养基的质量也是非常关键的，为此，我们提出了一种羊水细胞培养基及其制备方法。
发明内容
发明的目的在于提供一种羊水细胞培养基及其制备方法，以解决上述背景技术中提出的问题。
为实现上述目的，发明提供如下技术方案：一种羊水细胞培养基，其特征在于：以基础培养基的体积作为基准，所述添加成分组成及其含量分别为结合蛋白，10-15mg/L、转铁蛋白，10-15mg/L、胰岛素，15-20mg/L、L-谷氨酰胺，2-4mM、大豆胰酶，2-4mg/L、生长因子，5-15ug/L、孕酮，1-2nmol/L、氢化皮质酮，2-3nmol/L、葡萄糖，5-25mM、脂类复合物，2-4mM以及亚硒酸钠，20-40nmol/L。
优选的，所述基础培养基采用DMEM/F12(1：1，V/V)基础培养基。
优选的，所述DMEM/F12(1：1，V/V)基础培养基另添加有15-20mmol/LHEPES作为氢离子缓冲剂。
优选的，所述DMEM/F12(1：1，V/V)基础培养基另添加有10-20mg/L维生素E、10-20mg/LL-抗坏血酸作为抗氧化剂。
优选的，一种羊水细胞培养基的制备方法，包括以下步骤：
S1：DMEM/F12(1：1，V/V)基础培养基用900mL的注射水溶解后，添加碳酸氢钠1.2g和HEPES15-20mmol/L，混匀后静置；
S2：往S1溶解的DMEM/F12(1：1，V/V)基础培养基中添加结合蛋白10-15mg/L、转铁蛋白10-15mg/L、胰岛素15-20mg/L、L-谷氨酰胺2-4mM、大豆胰酶2-4mg/L、生长因子5-15ug/L、孕酮1-2nmol/L、氢化皮质酮2-3nmol/L、葡萄糖5-25mM、脂类复合物2-4mM以及亚硒酸钠20-40nmol/L后，静置；
S3：往S2所得溶液中添加10-20mg/L维生素E、10-20mg/LL-抗坏血酸后，添加注射水定容至1L，置于-20℃条件下保存即可。
优选的，所述置于-20℃保存之前用0.2μm滤膜进行过滤除菌。
优选的，所述静置时间不低于30min。
与现有技术相比，发明的有益效果是：
1、该种羊水细胞培养基，采用无血清等动物源的成分，排除血清中含有许多不确定物质(如不同的抗原、抗体、激素和细胞因子等)对于羊水细胞体外培养造成的不利影响，同时该羊水细胞培养基添加有各种营养物质能够很好的供羊水细胞体外扩增过程中一系列的生命活动，进而可以代替牛血清等血成分对于羊水细胞增殖过程中所起的作用，保证羊水细胞可进行稳定的体外增殖过程。
2、该种羊水细胞培养基，添加有维生素E和L-抗坏血酸作为抗氧化剂能够促进羊水细胞的成长，具有协同效应，使得羊水细胞体外培养过程中克隆总数、增殖速率等均有一定的提高，进而能够大大缩短羊水细胞的培养周期，并能够通过染色体显带技术，能够辨别三体综合征、平衡易位、倒位及性染色体异常等染色体方面的疾病。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例一：
发明提供一种技术方案：一种羊水细胞培养基，包括基础培养基以及添加在所述基础培养基中的添加成分，其特征在于：以DMEM/F12(1：1，V/V)基础培养基的体积作为基准，所述添加成分组成及其含量分别为结合蛋白，12.5mg/L、转铁蛋白，12.5mg/L、胰岛素，17.5mg/L、L-谷氨酰胺，3mM、大豆胰酶，3mg/L、生长因子，10ug/L、孕酮，1.5nmol/L、氢化皮质酮，2.5nmol/L、葡萄糖，10mM、脂类复合物，3mM以及亚硒酸钠，30nmol/L，另添加有20mmol/LHEPES作为氢离子缓冲剂以及20mg/L维生素E、20mg/LL-抗坏血酸作为抗氧化剂。
根据一种羊水细胞培养基的组成成分以及含量提供一种羊水细胞培养基的制备方法，包括以下步骤：
S1：DMEM/F12(1：1，V/V)基础培养基用900mL的注射水溶解后，添加碳酸氢钠1.2g和HEPES20mmol/L，混匀后静置30min以上；
S2：往S1溶解的DMEM/F12(1：1，V/V)基础培养基中添加结合蛋白，12.5mg/L、转铁蛋白，12.5mg/L、胰岛素，17.5mg/L、L-谷氨酰胺，3mM、大豆胰酶，3mg/L、生长因子，10ug/L、孕酮，1.5nmol/L、氢化皮质酮，2.5nmol/L、葡萄糖，10mM、脂类复合物，3mM以及亚硒酸钠，30nmol/L后，静置30min以上；
S3：往S2所得溶液中添加15mg/L维生素E、15mg/LL-抗坏血酸后，添加注射水定容至1L，用0.2μm滤膜进行过滤除菌后，置于-20℃条件下保存即可。
实施例二：
发明提供一种技术方案：一种羊水细胞培养基，包括基础培养基以及添加在所述基础培养基中的添加成分，其特征在于：以DMEM/F12(1：1，V/V)基础培养基的体积作为基准，所述添加成分组成及其含量分别为结合蛋白，10mg/L、转铁蛋白，10mg/L、胰岛素，15mg/L、L-谷氨酰胺，2mM、大豆胰酶，2mg/L、生长因子，5ug/L、孕酮，1nmol/L、氢化皮质酮，2nmol/L、葡萄糖，5mM、脂类复合物，2mM以及亚硒酸钠，20nmol/L，另添加有15mmol/LHEPES作为氢离子缓冲剂以及10mg/L维生素E、10mg/LL-抗坏血酸作为抗氧化剂。
根据一种羊水细胞培养基的组成成分以及含量提供一种羊水细胞培养基的制备方法，包括以下步骤：
S1：DMEM/F12(1：1，V/V)基础培养基用900mL的注射水溶解后，添加碳酸氢钠1.2g和HEPES15mmol/L，混匀后静置30min以上；
S2：往S1溶解的DMEM/F12(1：1，V/V)基础培养基中添加结合蛋白，10mg/L、转铁蛋白，10mg/L、胰岛素，15mg/L、L-谷氨酰胺，2mM、大豆胰酶，2mg/L、生长因子，5ug/L、孕酮，1nmol/L、氢化皮质酮，2nmol/L、葡萄糖，5mM、脂类复合物，2mM以及亚硒酸钠，20nmol/L后，静置30min以上；
S3：往S2所得溶液中添加10mg/L维生素E、10mg/LL-抗坏血酸后，添加注射水定容至1L，用0.2μm滤膜进行过滤除菌后，置于-20℃条件下保存即可。
实施例三：
发明提供一种技术方案：一种羊水细胞培养基，包括基础培养基以及添加在所述基础培养基中的添加成分，其特征在于：以DMEM/F12(1：1，V/V)基础培养基的体积作为基准，所述添加成分组成及其含量分别为结合蛋白，15mg/L、转铁蛋白，15mg/L、胰岛素，20mg/L、L-谷氨酰胺，4mM、大豆胰酶，4mg/L、生长因子，15ug/L、孕酮，2nmol/L、氢化皮质酮，3nmol/L、葡萄糖，25mM、脂类复合物，4mM以及亚硒酸钠，40nmol/L，另添加有15mmol/LHEPES作为氢离子缓冲剂以及10mg/L维生素E、10mg/LL-抗坏血酸作为抗氧化剂。
根据一种羊水细胞培养基的组成成分以及含量提供一种羊水细胞培养基的制备方法，包括以下步骤：
S1：DMEM/F12(1：1，V/V)基础培养基用900mL的注射水溶解后，添加碳酸氢钠1.2g和HEPES15mmol/L，混匀后静置30min以上；
S2：往S1溶解的DMEM/F12(1：1，V/V)基础培养基中添加结合蛋白，10mg/L、转铁蛋白，10mg/L、胰岛素，15mg/L、L-谷氨酰胺，2mM、大豆胰酶，2mg/L、生长因子，5ug/L、孕酮，1nmol/L、氢化皮质酮，2nmol/L、葡萄糖，5mM、脂类复合物，2mM以及亚硒酸钠，20nmol/L后，静置30min以上；
S3：往S2所得溶液中添加20mg/L维生素E、20mg/LL-抗坏血酸后，添加注射水定容至1L，用0.2μm滤膜进行过滤除菌后，置于-20℃条件下保存即可。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (7)
1.一种羊水细胞培养基，包括基础培养基以及添加在所述基础培养基中的添加成分，其特征在于：以基础培养基的体积作为基准，所述添加成分组成及其含量分别为结合蛋白，10-15mg/L、转铁蛋白，10-15mg/L、胰岛素，15-20mg/L、L-谷氨酰胺，2-4mM、大豆胰酶，2-4mg/L、生长因子，5-15ug/L、孕酮，1-2nmol/L、氢化皮质酮，2-3nmol/L、葡萄糖，5-25mM、脂类复合物，2-4mM以及亚硒酸钠，20-40nmol/L。
2.如权利要求1所述的一种羊水细胞培养基，其特征在于：所述基础培养基采用DMEM/F12(1：1，V/V)基础培养基。
3.如权利要求1或2所述的一种羊水细胞培养基，其特征在于：所述DMEM/F12(1：1，V/V)基础培养基另添加有15-20mmol/LHEPES作为氢离子缓冲剂。
4.如权利要求1或2所述的一种羊水细胞培养基，其特征在于：所述DMEM/F12(1：1，V/V)基础培养基另添加有10-20mg/L维生素E、10-20mg/LL-抗坏血酸作为抗氧化剂。
5.如权利要求1-4任一所述的一种羊水细胞培养基的制备方法，包括以下步骤：
S1：DMEM/F12(1：1，V/V)基础培养基用900mL的注射水溶解后，添加碳酸氢钠1.2g和HEPES15-20mmol/L，混匀后静置；
S2：往S1溶解的DMEM/F12(1：1，V/V)基础培养基中添加结合蛋白10-15mg/L、转铁蛋白10-15mg/L、胰岛素15-20mg/L、L-谷氨酰胺2-4mM、大豆胰酶2-4mg/L、生长因子5-15ug/L、孕酮1-2nmol/L、氢化皮质酮2-3nmol/L、葡萄糖5-25mM、脂类复合物2-4mM以及亚硒酸钠20-40nmol/L后，静置；
S3：往S2所得溶液中添加10-20mg/L维生素E、10-20mg/LL-抗坏血酸后，添加注射水定容至1L，置于-20℃条件下保存即可。
6.如权利要求5所述的一种羊水细胞培养基及其制备方法，其特征在于：所述置于-20℃保存之前用0.2μm滤膜进行过滤除菌。
7.如权利要求5所述的一种羊水细胞培养基及其制备方法，其特征在于：所述静置时间不低于30min。
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